Fiber-coupled, high-speed, integrated, angled-dual-axis confocal scanning microscopes employing vertical cross-section scanning

ABSTRACT

This invention provides an angled-dual-axis confocal scanning microscope comprising a fiber-coupled, angled-dual-axis confocal scanning head and a vertical scanning unit. The angled-dual-axis confocal scanning head is configured such that an illumination beam and an observation beam intersect optimally at an angle θ within an object and the scanning is achieved by pivoting the illumination and observation beams using a single scanning element, thereby producing an arc-line scan. The vertical scanning unit causes the angled-dual-axis confocal scanning head to move towards or away from the object. By integrating the angled-dual-axis confocal scanning microscope of the present invention with fiber-optic components and a fiber-coupled laser, the present invention also provides an assembly of fiber-based angled-dual-axis confocal scanning systems that can be particularly powerful tools in biological and medical imaging applications, such as instruments for performing optical coherence microscopy and in vivo optical biopsies.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part application of a U.S. patentapplication Ser. No. 09/628,118, filed on Jul. 28, 2000, now allowed.This application also relates to the following U.S. Patent Applications,all of which are hereby incorporated herein by reference: Ser. No.09/627,363, filed on Jul. 28, 2000, U.S. Pat. No. 6,351,325; Ser. No.09/628,119, filed on Jul. 28, 2000, now allowed; Ser. No. 09/728,566,filed on Nov. 30, 2000, U.S. Pat. No. 6,414,779; and Ser. No.09/705,284, filed on Nov. 1, 2000, U.S. Pat. No. 6,369,928.

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of confocalmicroscopes, and in particular, to a new class of fiber-coupled,angled-dual-axis confocal scanning microscopes with integratedstructure, enhanced resolution, low noise, and vertical cross-sectionscanning.

BACKGROUND ART

[0003] The advent of fiber optics and laser technology has brought arenewed life to many areas of conventional optics. Confocal microscopes,for example, have enjoyed higher resolution, more integrated structure,and enhanced imaging capability. Consequently, confocal microscopes havebecome increasingly powerful tools in a variety of applications,including biological and medical imaging, optical data storage andindustrial applications.

[0004] The original idea of confocal microscopy traces back to the workof Marvin Minsky. Described in his seminal U.S. Pat. No. 3,013,467 is asystem in which an illumination beam passes through a pinhole, traversesa beamsplitter, and is focused by an objective to a focal volume withinan object. An observation beam that emanates from the focal volume is inturn converged by the same objective lens, reflected by its secondencounter with the beamsplitter, and passes through a second pinhole toan optical detector. The geometry of this confocal arrangement is suchthat only the light beam originating from the focal volume is able topass through the second pinhole and reach the optical detector, thuseffectively discriminating all other out-of-focus signals.

[0005] Contemporary confocal microscopes tend to adopt one of two basicconfocal geometries. In the transmission arrangement using twoobjectives, one objective focuses an illumination beam from a pointsource onto a focal volume within an object and another objectivecollects an observation beam that emanates from the focal volume.Whereas in the so-called “reciprocal” reflection arrangement, a singleobjective plays a dual role of focusing light on the object andcollecting the light emanated from the object. In either case, theconfocal arrangement enables the confocal microscope to attain a higherresolution and sharper definition than a conventional microscope,because out-of-focus signals are rejected. This unique ability has madeconfocal microscopes particularly useful tools in the examination ofbiological specimens, since they can view a specific layer within asample and avoid seeing other layers, the so-called “opticalsectioning”.

[0006] In order to image a thin layer about a few micrometers thickwithin a sample, however, the numerical aperture (NA) of the objectivelenses must be large, so as to provide adequate resolution particularlyin the axial direction. This generally results in a short workingdistance, which is undesirable in practice. Moreover, when imagingwithin tissue or scattering media, the signal is typically dominated byscattering from points far away from the focus of the large NAobjective, thus resulting in noisy (low contrast) images.

[0007] A great deal of ingenuity has accordingly been devoted toimproving the axial resolution of confocal microscopes without usinghigh NA lenses. A particularly interesting approach is to spatiallyarrange two separate illumination and observation objective lenses, orillumination and observation beam paths, in such a way that theillumination beam and the observation beam intersect at an angle theta(θ) at the focal points, so that the overall point-spread function forthe microscope, i.e., the overlapping volume of the illumination andobservation point-spread functions results in a substantial reduction inthe axial direction. A confocal microscope with such an angled,dual-axis design is termed a confocal theta microscope, or anangled-dual-axis confocal microscope, hereinafter. Its underlying theoryis stated below for the purpose of elucidating the principle of thisinvention. A more detailed theory of confocal theta microscopy can befound in U.S. Pat. No. 5,973,828; by Webb et al. in “Confocal microscopewith large field and working distance”, Applied optics, Vol.38, No.22,pp.4870; and by Stelzer et al. in “A new tool for the observation ofembryos and other large specimens: confocal theta fluorescencemicroscopy”, Journal of Microscopy, Vol.179, Part 1, pp. 1; allincorporated by reference.

[0008] The region of the point-spread function of a microscope'sobjective that is of most interest is the region in which thepoint-spread function reaches its maximum value. This region is referredto as the “main lobe” of the point-spread function in the art. It istypically characterized in three dimensions by an ellipsoid, whichextends considerably further in the axial direction than in thetransverse direction. This characteristic shape is the reason that theaxial resolution is inherently poorer than the transverse resolution ina conventional confocal microscope. When the main lobes of theillumination and observation point-spread functions are arranged tointersect at an angle in a confocal theta microscope, however, apredominantly transverse and therefore narrow section from one main lobeis made to multiply (i.e., zero out) a predominantly axial and thereforelong section from the other main lobe. This optimal multiplication ofthe two point-spread functions reduces the length of the axial sectionof the overall point-spread function, thereby enhancing the overallaxial resolution. The shape of the overall point-spread function can befurther adjusted by varying the angle at which the main lobes of theillumination and observation point-spread functions intersect.

[0009] The past few years have seen a few confocal theta microscopeswith similar designs in the art. For example, Stelzer et al. describethe theory of confocal theta microscopy with two and three objectivehigh NA lenses and an angle of θ=90° in “Fundamental reduction of theobservation volume in far-field light microscopy by detection orthogonalto the illumination axis: confocal theta microscopy”, OpticsCommunications 111, pp.536. German Patent DE-OS 43 26 473 A1demonstrates a confocal theta microscope in which the axes of two highNA objective lenses are oriented at a right angle (θ=90°). It alsodiscloses a confocal theta microscope with three high NA objectivelenses, in which the axes of two objectives are perpendicular to eachother, while the axis of the third objective lies on the axis of one ofthe other two objectives. The patent does not disclose how scanning iscarried out in these confocal theta microscopes. Although scanning mightbe performed by translating the object to be examined in such systems,the designs of these confocal theta microscopes are such that they donot appear to readily accommodate scanning that probes into the interiorof the object. U.S. Pat. No. 5,969,854 discloses a confocal thetamicroscope with the axes of two high NA objective lenses positioned atan angle approximately 90°. This system incorporates a scanningmechanism that translates the object for imaging purposes.

[0010] Webb et al. describe a confocal scanning microscope with angledobjective lenses that have relatively low NA in “Confocal microscopewith large field and working distance”, Applied Optics, Vol.38, No.22,pp.4870. The design of this microscope attains usable resolutions forbiological applications in both transverse and axial directions, whileachieving a large field of view and a long working distance. Thescanning in this case is achieved by moving a stage on which the objectis mounted. U.S. Pat. No. 5,973,828 discloses a confocal theta scanningmicroscope in which the axes of two objective lenses intersect at avariable angle θ. Two-dimensional scanning is achieved by steering theillumination and observation beams in the back focal plane of theobjective lenses to provide scanning in one direction, and by separatelymoving and coordinating the illumination and observation lenses to bringabout overlap of the focal volumes during scanning in the otherdirection. It is disclosed that without such coordination the overlapcannot be maintained throughout the scanning. U.S. Pat. No. 6,064,518describes a confocal theta microscope that uses one objective lens forfocusing the illumination beam onto an object and the same objective forcollecting the observation beam reflected from the object. This systememploys a beam deflection unit for directing the illumination beam fromthe objective lens onto an object and for passing the observation beamreflected from the object to the same objective lens. The patent pointsout that the scanning may be obtained by either moving the beamdeflection unit such that the illumination and observation beams scanthe object, or by moving the object itself via a translation stage.However, no specific scanning mechanism is disclosed. The design of thissystem is such that it does not lend itself to miniaturization and fastscanning as required for in vivo imaging applications. Moreover, itprovides inadequate working distance for in vivo imaging of livespecimens since the object must be placed between the objective and thedeflection unit.

[0011] One drawback to translating the object to be scanned is that inmany applications it is difficult, if not entirely impossible, tomaneuver the object such that high speed and precision scanning isattained. This problem can be particularly acute in imaging objects inscattering media, such as in vivo imaging of live tissue in biologicaland medical applications. Moving the two objective lenses throughseparate mechanisms as described in U.S. Pat. No. 5,973,828, on theother hand, requires that the translations of the illumination andobservation lenses be coordinated and synchronized such that the mainlobes of the illumination and observation point-spread functionsintersect optimally at all target points on the path to be scanned. Suchcoordination can be quite cumbersome to implement, rendering fast andhigh precision scanning difficult to achieve. Although the possibilityof scanning the illumination and observation beams is proposed in U.S.Pat. No. 6,064,518, the particular design of the confocal thetamicroscope disclosed in this patent does not lend itself to fast andmaneuverable scanning using this scheme.

[0012] Furthermore, as described in the above prior art, confocal thetamicroscopes use various mechanical pinholes to provide a point lightsource and a point detector. The disadvantage with using mechanicalpinholes is the lack of flexibility and ruggedness. The optical systemsincorporating mechanical pinholes are also difficult to align andminiaturize. In addition, slight misalignment of a mechanical pinhole orany other optical element can result in asymmetric intensitydistribution of the light emerging from the pinhole, causingaberrations.

[0013] In recent years optical fibers have been used in confocal systemsto transmit light more flexibly. A single-mode fiber is typically usedso that an end of the fiber is also conveniently utilized as a pinholefor both light emission and detection. This arrangement is notsusceptible to the alignment problems the mechanical pinholes in theprior art systems tend to suffer. Moreover, the use of optical fibersenables the microscopes to be more flexible and compact in structure,along with greater maneuverability in scanning. U.S. Pat. Nos.5,120,953, 5,161,053, 5,742,419 and 5,907,425, for instance, discloseconventional reciprocal confocal scanning microscopes using a singleoptical fiber to transmit light. The end of the fiber provides a pointillumination source and a point detector. The scanning in these systemsis achieved by maneuvering the fiber end. Confocal theta microscopy isnot employed in these systems, however.

[0014] Hence, what is needed in the art is a confocal scanningmicroscope that attains enhanced axial resolution, a long workingdistance and a large field of view, fast and high precision scanning,without involving complicated coordination of scanning actions. Thedesired confocal scanning microscope should also achieve highsensitivity and large dynamic range when imaging in a scattering medium,so as to produce high image contrast. Additionally, the desired confocalscanning microscope should have an integrated and scalable structure,rendering it a modular and versatile device.

OBJECTS AND ADVANTAGES

[0015] Accordingly it is a principal object of the present invention toprovide an angled-dual-axis confocal scanning microscope for imaging intissue or a scattering medium that:

[0016] a) uses low NA and therefore inexpensive objective lenses;

[0017] b) attains improved axial resolution;

[0018] c) provides a larger field of view;

[0019] d) affords a longer working distance;

[0020] e) achieves higher sensitivity and larger dynamic range ofdetection;

[0021] f) is fiber-coupled;

[0022] g) has higher power efficiency;

[0023] h) performs vertical cross-section scanning; and

[0024] i) has small, compact, integrated, and simple construction.

[0025] It is a further object of the present invention to provide anassembly of fiber-based angled-dual-axis confocal scanning systems thatadvantageously combine the angled-dual-axis confocal scanning microscopeof the present invention and fiber-optic components.

[0026] These and other objects and advantages will become apparent fromthe following description and accompanying drawings.

SUMMARY OF THE INVENTION

[0027] This invention provides an angled-dual-axis confocal scanningmicroscope, comprising an angled-dual-axis confocal scanning head and avertical scanning unit. The angled-dual-axis confocal scanning headfurther comprises a first end of a first single-mode optical fiberserving as a point light source, an angled-dual-axis focusing means, ascanning means, and a first end of a second single-mode optical fiberserving as a point light detector.

[0028] From the first end of the first optical fiber an illuminationbeam emerges. The angled-dual-axis focusing means serves to focus theillumination beam to a diffraction-limited illumination focal volumealong an illumination axis within an object. The angled-dual-axisfocusing means further receives an observation beam emanated from anobservation focal volume along an observation axis within the object,and focuses the observation beam to the first end of the second opticalfiber. The angled-dual-axis focusing means is so designed that theillumination axis and the observation axis intersect at an angle θwithin the object, such that the illumination and observation focalvolumes intersect optimally at a confocal overlapping volume. Thescanning means, in the form of a single scanning element disposedbetween the angled-dual-axis focusing means and the object, ispositioned such that it receives the illumination beam from theangled-dual-axis focusing means and directs the illumination beam to theobject; and that it collects the observation beam emanated from theobject and passes the observation beam to the angled-dual-axis focusingmeans. The scanning means is further capable of pivoting theillumination and observation beams jointly in such a way that theillumination and observation axes remain intersecting optimally at afixed angle θ and that the confocal overlapping volume moves along anarc-line within the object, thereby producing an arc-line scan. Thevertical scanning unit comprises a vertical translation means and acompensation means. The vertical translation means is mechanicallycoupled to the angled-dual-axis confocal scanning head, such that itcauses the angled-dual-axis confocal scanning head to move towards oraway from the object, whereby a succession of arc-line scans thatprogressively deepen into the object is produced, providing atwo-dimensional vertical cross-section scan of the object. Thecompensation means keeps the optical path lengths of the illuminationand observation beams substantially unchanged, thereby ensuring theoptimal intersection of the illumination and observation focal volumesin the course of vertical scanning. Altogether, the angled-dual-axisconfocal scanning microscope of the present invention is designed suchthat it is capable of performing vertical cross-section scanning in aline-by-line fashion with enhanced axial (i.e., vertical) resolution andgreater speed, while maintaining a workable working distance and a largefield of view. Additionally, the object may be moved incrementally in adirection perpendicular to the vertical cross-section scan plane as eachvertical cross-section scan is completed, resulting in a plurality ofvertical cross-section images that can be assembled into athree-dimensional image of a region within the object.

[0029] It is to be understood that the term “emanating” as used in thisspecification is to be construed in a broad sense as covering any lighttransmitted back from the object, including reflected light, scatteredlight, and fluorescent light. It should be also understood that whendescribing the intersection of the illumination and observation beams inthis specification, the term “optimal” means that the illumination andobservation focal volumes (i.e., the main lobes of the illuminationbeam's point-spread function and the observation beam's point-spreadfunction) intersect in such a way that their respective centerssubstantially coincide and the resulting overlapping volume hascomparable transverse and axial extents. This optimal overlapping volumeis termed “confocal overlapping volume” in this specification.

[0030] In an angled-dual-axis confocal scanning head of the presentinvention, the angled-dual-axis focusing means generally comprises anassembly of beam focusing, collimating, and deflecting elements. Suchelements can be selected from the group of refractive lenses,diffractive lenses, GRIN lenses, focusing gratings, micro-lenses,holographic optical elements, binary lenses, curved mirrors, flatmirrors, prisms and the like. A crucial feature of the angled-dual-axisfocusing means is that it provides an illumination axis and anobservation axis that intersect optimally at an angle θ. The scanningmeans typically comprises an element selected from the group consistingof mirrors, reflectors, acousto-optic deflectors, electro-opticdeflectors, mechanical scanning mechanisms, andMicro-Electro-Mechanical-Systems (MEMS) scanning micro-mirrors. A keyfeature is that the scanning means is in the form of a single element,as opposed to two or more separate scanning elements in many prior artconfocal scanning systems. A preferred choice for the scanning means isa flat pivoting mirror, particularly a silicon micro-machined scanningmirror for its compact and light-weight construction. Moreover, theoptical fibers can be single-mode fibers, multi-mode fibers, birefrigentfibers, polarization maintaining fibers and the like. Single-mode fibersare preferable in the present invention, for the ends of single-modefibers provide a nearly point-like light source and detector.

[0031] A unique feature of the angled-dual-axis confocal scanning headof the present invention is that the scanning means is placed betweenthe angled-dual-axis focusing means and the object to be examined. Thisenables the best on-axis illumination and observation point-spreadfunctions to be utilized throughout the entire angular range of anarc-line scan, thereby providing greater resolution over a largertransverse field of view, while maintaining diffraction-limitedperformance. Such an arrangement is made possible by taking advantage ofthe longer working distance rendered by using relatively lower NAfocusing elements or lenses in the angled-dual-axis focusing means.

[0032] Another important advantage of the angled-dual-axis arrangementof the present invention is that since the observation beam ispositioned at an angle relative to the illumination beam, scatteredlight along the illumination beam does not easily get passed into theobservation beam, except where the beams overlap. This substantiallyreduces scattered photon noise in the observation beam, thus enhancingthe sensitivity and dynamic range of detection. This is in contrast tothe direct coupling of scattered photon noise between the illuminationand observation beams in a transmission or reciprocal confocalmicroscope, due to the collinear arrangement between the beams.Moreover, by using low NA focusing elements (or lenses) in anangled-dual-axis confocal scanning system of the present invention, theillumination and observation beams do not become overlapping until veryclose to the focus. Such an arrangement further prevents scattered lightin the illumination beam from directly “jumping” to the observationbeam, hence further filtering out scattered photon noise in theobservation beam. Altogether, the angled-dual-axis confocal system ofthe present invention has much lower noise and is capable of providingmuch higher contrast when imaging in a scattering medium than the priorart confocal systems employing high NA lenses, rendering it highlysuitable for imaging within biological specimens.

[0033] A further advantage of the present invention is that the entireangled-dual-axis confocal scanning head can be mounted on a siliconsubstrate etched with precision V-grooves where various optical elementsare hosted. Such an integrated device offers a high degree of integrity,maneuverability, scalability, and versatility, while maintaining aworkable working distance and a large field of view. In particular, amicro-optic version of an integrated, angled-dual-axis confocal scanninghead of the present invention can be very useful in biological andmedical imaging applications, e.g., endoscopes and hand-held opticalbiopsy instruments.

[0034] The present invention further provides a first angled-dual-axisconfocal scanning system, comprising an angled-dual-axis confocalscanning microscope of the present invention, a light source, and anoptical detector. The light source is optically coupled to the secondend of the first optical fiber of the angled-dual-axis confocal scanningmicroscope, providing an illumination beam; and the optical detector isoptically coupled to a second end of the second optical fiber of theangled-dual-axis confocal scanning microscope, receiving an observationbeam collected from an object. The light source can be a continuous wave(CW) or a pulsed source such as a fiber laser, a semiconductor opticalamplifier, an optical fiber amplifier, a semiconductor laser, a diodepumped solid state laser, or other suitable fiber-coupled light sourceknown in the art. The optical detector can be a PIN diode, an avalanchephoto diode (APD), or a photomultiplier tube. Such an angled-dual-axisconfocal scanning system provides a simple and versatile imaging toolwith high resolution and fast scanning capability.

[0035] It is known in the art that many biological tissues, such astendons, muscle, nerve, bone, cartilage and teeth, exhibit birefrigencedue to their linear or fibrous structure. Birefrigence causes thepolarization state of light to be altered (e.g., rotated) in aprescribed manner upon refection. Skin is another birefrigent medium.Collagen contained in skin is a weakly birefrigent material. Attemperatures between 56-65° C., collagen denatures and loses its.birefrigence. Thus, by detecting induced changes in the polarizationstate of light reflected from a skin sample, an image representing theregions of skin where thermal injury occurs can be identified. Theangled-dual-axis confocal scanning system described above can bemodified to image such a birefrigent-scattering (or otherpolarization-altering) medium. A polarized light source is opticallycoupled to a second end of the first optical fiber of theangled-dual-axis confocal scanning microscope, providing a polarizedillumination beam. The birefrigent (or other polarization-altering)“scatterers” emanate an observation beam whose polarization is altered(e.g.,, rotated) relative to the polarization of the illumination beam.Such a rotated polarization can be represented in two orthogonalpolarization components. A polarizing beamsplitter is then opticallycoupled to a second end of the second optical fiber of theangled-dual-axis confocal scanning microscope, serving to route the twoorthogonal polarization components of the observation beam to twoseparate optical detectors. An image representing the birefrigent (orother polarization-altering) “scatterers” can be accordinglyconstructed.

[0036] The present invention also provides an angled-dual-axis confocalscanning module, comprising an angled-dual-axis confocal scanningmicroscope of the present invention optically coupled to anon-reciprocal three-port optical circulator. The third and first portsof the optical circulator are optically coupled to the second ends ofthe first and second optical fibers of the angled-dual-axis confocalscanning microscope, respectively; and the second port of the opticalcirculator serves as a bi-directional input/output port. Theconfiguration of the angled-dual-axis confocal scanning module is suchthat an illumination beam transmitted to the second port is in turnpassed into the third port of the optical circulator and then coupled tothe second end of the first optical fiber of the angled-dual-axisconfocal scanning microscope in nearly its entirety; and an observationbeam collected by the angled-dual-axis confocal scanning microscope isdelivered to the first port and-then routed to the second port of theoptical circulator, to be further utilized or detected in nearly itsentirety. As such, the angled-dual-axis confocal scanning module of thepresent invention provides a modular angled-dual-axis confocal scanningdevice with a single input/output port, and can be readily adapted in avariety of applications, as the following embodiments demonstrate.

[0037] For example, by coupling the angled-dual-axis confocal scanningmodule of the present invention to a first output aperture of aself-detecting laser source having two output apertures, an illuminationbeam is transmitted from the first output aperture of the laser to theangled-dual-axis confocal scanning module, and an observation beamcollected by the module is in turn back coupled to the laser via thesame output aperture. The feedback of the observation beam emanated froman object alters the light intensity as well as the modes supported bythe laser cavity, and the resulting changes or perturbations can bedetected by coupling an optical detector to a second output aperture ofthe laser. The presence of the non-reciprocal optical circulator in theangled-dual-axis confocal scanning module allows nearly 100% of theobservation beam to be back coupled to the laser, hence maximizing thesignal-to-noise ratio in detection. The use of a self-detecting laser asan integrated light source and detector further simplifies the structureof this angled-dual-axis confocal scanning system. Moreover, a frequencyshifter (or a phase modulator) can be optically coupled to thisangled-dual-axis confocal scanning system, arranged such that thefrequency of the observation beam is shifted. The feedback of thefrequency-shifted (or phase-modulated) observation beam to the laserresults in the laser's output beam being modulated at a beat frequency,thereby allowing for more sensitive heterodyne detection. The systemthus described constitutes the second angled-dual-axis confocal scanningsystem of the present invention.

[0038] If the self-detection laser source is equipped with only oneoutput aperture, the angled-dual-axis confocal scanning module of thepresent invention can be optically coupled to the laser via abeam-splitting means, such as a 90/10 fiber-optic coupler or otherlow-coupling tap coupler. The beam-splitting means serves to divert aportion of the laser's output beam, which carries the perturbations dueto the back coupling of the observation beam, to a detection path towhich an optical detector may be coupled. Such a system constitutes thethird angled-dual-axis confocal scanning system of the presentinvention.

[0039] The self-detecting characteristics of lasers have beenadvantageously exploited in the art to provide an integrated lightsource and detector, which also demonstrates the inherent highsensitivity of this method of optical detection. A great deal of efforthas also been devoted to eliminate such sensitive feedback effects(e.g., optical isolators with non-reciprocal optical elements such asFaraday rotators are designed to eliminate or block the back-coupling oflight). In the present invention, the self-detecting laser can be afiber laser, a semiconductor laser, or a diode pumped solid state laser.A fiber-based laser system, such as the fiber laser disclosed by theinventors of this application in U.S. Pat. No. 5,887,009, may be used totake advantage of the inherent flexibility of laser cavity parameters. Asemiconductor laser may also be desirable as a low cost device.

[0040] The angled-dual-axis confocal scanning module of the presentinvention can also be optically coupled to a light source via a secondnon-reciprocal, three-port optical circulator. In this embodiment, anoutput aperture of the light source is optically coupled to a first portof the second optical circulator and a second port of the second opticalcirculator is in turn optically coupled to the input/output port of theangled-dual-axis confocal scanning module, such that an illuminationbeam is passed from the light source into the angled-dual-axis confocalscanning module in nearly its entirety. The optical coupling between thesecond optical circulator and the angled-dual-axis confocal scanningmodule is preferably provided by a single optical fiber, though otheroptical coupling means can also be implemented. An observation beamcollected by the angled-dual-axis confocal scanning module is thenrouted to a third port of the second optical circulator, which furtherleads to a detection path, preferably in the form of a detection opticalfiber. An optical detector may be optically coupled to the detectionoptical fiber. In this angled-dual-axis confocal scanning system, thelight source may be any suitable laser or non-laser source, whichoperates in either continuous or pulsed mode. In fact, a skilled artisanmay implement any light source suitable for a given application.Moreover, the non-reciprocal nature of the second optical circulatorallows nearly 100% of the observation beam to be used for detection,hence maximizing the signal-to-noise ratio. The system thus describedconstitutes the fourth angled-dual-axis confocal scanning system of thepresent invention.

[0041] The fourth angled-dual-axis confocal scanning system describedabove can be further modified into an interferometer configuration, suchthat the observation beam is combined with a portion of the output beamfrom the light source to create coherent interference. This can beachieved by inserting a beam-splitting means, such as a fiber-opticcoupler or a beamsplitter, between the light source and the secondoptical circulator. In such an arrangement, the beamsplitting meansdiverts a portion of the output beam emitted from the light source tothe first port of the second optical circulator, which is in turn routedto the angled-dual-axis confocal scanning module, providing anillumination beam. The remainder of the output beam from the lightsource is diverted to a reference path, preferably in the form of areference optical fiber, providing a reference beam. The third port ofthe second optical circulator then routes an observation beam collectedby the angled-dual-axis confocal scanning module to a detection path,preferably in the form of a detection optical fiber. The reference anddetection optical fibers may be coupled by a 50/50 fiber-optic couplerto mix the observation and reference beams, and produce two outputs witha n phase difference for use in a balanced detection scheme. In thisway, an interferometer is created and the length of the referenceoptical fiber can be adjusted to achieve coherent interference betweenthe observation and reference beams.

[0042] The system described above, hence the fifth angled-dual-axisconfocal scanning system of the present invention, may further include afrequency shifter (or a phase modulator), arranged such that thefrequency of either the reference or the observation beam is shifted, soas to generate coherent heterodyne interference between the observationand reference beams. Heterodyne balanced detection technique, well-knownin the art of optical coherence tomography (OCT), can be accordinglyutilized. An adjustable optical delay device can also be implemented insuch a way to maintain coherent interference between the reference andobservation beams. If the light source has a short coherence length,then the delay can be adjusted such that only single-scattered light inthe observation beam is coherent with the reference beam at the 50/50fiber-optic coupler and multiple-scattered light, which traverses over alarger optical path length in the observation beam, does not contributeto the coherent interference, therefore providing further filtering ofmultiple-scattered light. To enhance the signal-to-noise ratio indetection, an optical amplifier, such as a two-port fiber amplifier orsemiconductor optical amplifier (SOA), can be coupled to the detectionoptical fiber, such that the observation beam is amplified. An amplifiedobservation beam also allows faster scanning rates and consequentlyhigher pixel rates without appreciable loss in signal-to-noise ratio,because a shorter integration time per pixel of an image is required indata collection.

[0043] The light source in the fifth angled-dual-axis confocal scanningsystem of the present invention can be an optical fiber amplifier, asemiconductor optical amplifier, a fiber laser, a semiconductor laser, adiode-pumped solid state laser, or a continuous wave or pulsed broadbandOCT source having a short coherence length, as is well known in the art.If polarized light is provided by the light source, the beam-splittingmeans should be a polarizing beamsplitter, such as a polarizingbeamsplitter evanescent wave optical fiber coupler, and the variousoptical fibers in the system should be polarization maintaining (PM)fibers. In this case, the observation and reference beams can be broughtinto the same polarization by rotation of either the reference ordetection optical fiber. Alternatively, a polarization rotation means,such as a Faraday rotator, can be coupled to either the reference ordetection optical fiber, such that the reference and observation beamshave substantially the same polarization when combined. Furthermore, the50/50 fiber-optic coupler can be a polarization maintaining fibercoupler to optimally mix the polarized observation and reference beams.

[0044] A distinct advantage of the angled-dual-axis confocal scanningmicroscope of the present invention is that the scanning is achieved bypivoting both the illumination and observation beams, as opposed tomoving either the object or the microscope's objective lenses in theprior art confocal theta scanning microscopes, which adversely limitsthe speed and maneuverability of scanning. Moreover, a single-elementscanning means, such as a micro-machined scanning mirror, is used topivot the illumination and observation beams jointly, in contrast to theprior art systems where the two beams are scanned individually by way ofmoving the microscope's objectives lenses separately, which requiresprecise synchronization and coordination in maneuvering the lenses. Inaddition, by disposing the scanning means between the angled-dual-axisfocusing means and the object, fast and high-precision scanning at highresolution is obtained over a large field of view. Such an arrangementtakes advantage of the long working distance rendered by using low NAfocusing elements (or lenses). Another important advantage gained byusing low NA focusing elements is that the illumination and observationbeams do not become overlapping until sufficiently close to the focus.This prevents scattered light in one beam from directly “jumping” toanother beam, hence eliminating scattered photon noise in theobservation beam. Furthermore, low NA lenses can be easily designed foraberration correction, thus allowing diffraction-limited performance atrelatively low cost. In the present invention, diffraction-limitedfocusing is only required “on-axis”, hence further simplifying the lensrequirements. The angled-dual-axis confocal scanning microscope of thepresent invention further advantageously exploits the flexibility,scalability and integrity afforded by optical fibers and siliconmicro-machining techniques, rendering it a highly versatile and modulardevice. Accordingly, the angled-dual-axis confocal scanning microscopeof the present invention is particularly suited for applications inwhich high resolution and fast scanning are required, such as in vivoimaging of live tissue for performing optical biopsies in medicalapplications.

[0045] By integrating the angled-dual-axis confocal scanning microscopeof the present invention with fiber-optic components and a fiber-coupledlaser, the angled-dual-axis confocal scanning systems of the presentinvention provide a diverse assembly of fiber-based, high resolution andfast scanning systems that can be adapted in a variety of applications,such as in biological and medical imaging, and industrial applications.

[0046] The novel features of this invention, as well as the inventionitself, will be best understood from the following drawings and detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

[0047] FIGS. 1A-1D shows several exemplary embodiments of anangled-dual-axis confocal scanning head according to the presentinvention;

[0048] FIGS. 2A-2B depict two exemplary embodiments of anangled-dual-axis confocal scanning microscope according to the presentinvention;

[0049] FIGS. 3A-3B show two exemplary embodiments of a firstangled-dual-axis confocal scanning system according to the presentinvention;

[0050]FIG. 4 depicts an exemplary embodiment of an angled-dual-axisconfocal scanning module according to the present invention;

[0051] FIGS. 5A-5B show simplified schematic diagrams of second andthird angled-dual-axis confocal scanning systems according to thepresent invention;

[0052] FIGS. 6A-6B depict simplified schematic diagrams of fourth andfifth angled-dual-axis confocal scanning systems according to thepresent invention;

[0053]FIG. 7 illustrates a configuration of two intersecting beamsaccording to an embodiment of the present invention; and

[0054] FIGS. 8A-8C show graphs of normalized confocal-signal as afunction of position in the x, y, z directions according to exemplaryembodiments of the present invention; and

[0055]FIG. 9 shows another exemplary embodiment of an angled-dual-axisconfocal scanning head according to the present invention.

DETAILED DESCRIPTION

[0056] Although the following detailed description contains manyspecific details for the purposes of illustration, anyone of ordinaryskill in the art will appreciate that many variations and alterations tothe following details are within the scope of the invention.Accordingly, the exemplary embodiment of the invention described belowis set forth without any loss of generality to, and without imposinglimitations upon, the claimed invention.

[0057] FIGS. 1A-1D depict several exemplary embodiments of anangled-dual-axis confocal scanning head according to the presentinvention. Depicted in FIG. 1A is a first exemplary embodiment of anangled-dual-axis confocal scanning head of the present invention.Confocal scanning head 100, by way of example, comprises a first end 101of a first optical fiber 103 serving as a point light source; a firstend 102 of a second optical fiber 104 serving as a point light detector;an angled-dual-axis focusing means in the form of a lens assemblyconsisting of first collimating lens 105, second collimating lens 106,illumination lens 107, observation lens 108, and two beam-aligningelements in the form of first 45-degree mirror 109 and second 45-degreemirror 110; a scanning means in the form of a single scanning mirror111, which can be pivoted about axis 122, and a silicon substrate 112.First collimating lens 105, illumination lens 107, and first mirror 109are mounted on first V-groove 113 etched on substrate 112. Similarly,second collimating lens 106, observation lens 108, and second mirror 110are mounted on second V-groove 114 etched on substrate 112. Firstoptical fiber 103 and second optical fiber 104 are affixed tocylindrical ferrules 115 and 116 respectively, which are in turn mountedon V-grooves 113 and 114 respectively, such that fiber ends 101 and 102are held in their respective positions.

[0058] In operation, an illumination beam 117 emerges from first end 101of first optical fiber 103 and is directed to first collimating lens105. The collimated beam is then passed onto and focused by illuminationlens 107. The focused beam is further deflected by first mirror 109 toscanning mirror 111, which in turn directs the beam to adiffraction-limited illumination focal volume (see FIG. 1B) within anobject 120. An observation beam 118 emanated from a diffraction-limited,confocal overlapping volume 121 is first collected by scanning mirror111, then directed to second mirror 110, which in turn deflects the beamto observation lens 108. Observation beam 118 is then collimated byobservation lens 108 and then focused by second collimating lens 106 tofirst end 102 of second optical fiber 104. Scanning mirror 111 ispositioned such that illumination beam 117 and observation beam 118intersect optimally at an angle (θ) within object 120.

[0059]FIG. 1B provides a more detailed illustration of how illuminationbeam 117 and observation beam 118 are arranged to intersect withinobject 120 in FIG. 1A. Illumination beam 117, directed by scanningmirror 111, is focused to an illumination focal volume 99 oriented alongan illumination axis 97 within object 120. Observation beam 118 emanatesfrom an observation focal volume 98 oriented along an observation axis96 within object 120. Observation beam 118 is received by scanningmirror 111. Illumination axis 97 and observation axis 96 are directed tointersect at an angle θ, such that illumination focal volume 99 andobservation focal volume 98 intersect optimally at confocal overlappingvolume 121. A three-dimensional x-y-z coordinate system is provided todescribe the spatial extents of confocal overlapping volume 121, wherethe origin of the coordinate system is set at the center of confocaloverlapping volume 121. The z-axis defines the axial (or vertical)direction, whereas x-axis and y-axis (pointing out of the page)represent two orthogonal transverse directions.

[0060] It is to be understood that the term “emanating” as used in thisspecification is to be construed in a broad sense as covering any lighttransmitted back from the object, including reflected light, scatteredlight, or fluorescent light. A skilled artisan will know how toselectively make use of a particular type of light collected from theobject and filter out spurious background light for a given application.

[0061] In the present invention, various optical elements areaberration-corrected, and single-mode optical fibers are used to providea point light source and detector. Accordingly, illumination focalvolume 99 and observation focal volume 98 described above arediffraction-limited, defined by the main lobes of the illuminationbeam's point-spread function and observation beam's point-spreadfunction. Confocal overlapping volume 121 is likewisediffraction-limited, determined by an optimal overlapping of the mainlobes of the illumination beam's point-spread function and theobservation beam's point-spread function, as illustrated in FIG. 1B.

[0062] The present invention provides a model for calculating thepoint-spread functions of two optimally intersecting focused beams, suchas illumination beam 117 and observation beam 118 exemplified in FIG.1B, thereby providing an estimate of the three-dimensional extents ofthe resulting confocal overlapping volume, such as confocal overlappingvolume 121. The model calculation is presented as follows.

[0063]FIG. 7 illustrates a configuration in which two focused,diffraction-limited beams beam-1, beam-2, intersect at an angle 2α,where beam-1 and beam-2 can be illumination and observation beamsrespectively in an angled-dual-axis arrangement described above. Acentral x-y-z coordinate system is provided, such that z-axis definesthe axial (or vertical) direction, and x-axis and y-axis (pointing outof the page) represent two orthogonal transverse directions. There aretwo additional coordinate systems, where the x₁-y₁-z₁ coordinate systemis associated with beam-1 and the x₂-y₂-z₂ coordinate system isassociated with beam-2. All three coordinate systems share the sameorigin O, which is located at the center of the two-beam overlappingregion. The transformations between the coordinate systems associatedwith the beams and the central-coordinate system are given by:

x ₁ =x cos α+z sin α

y₁=y

z ₁ =−x sin α+z cos α

x ₂ =x cos α−z sin α

y₂=y

z ₂ =x sin α+z cos α

[0064] Using paraxial theory, and assuming that the electric-fieldassociated with each beam is polarized in the y direction (i.e.,perpendicular to the page). (It can be shown that for reflection from aperfect mirror or from a small particle, the same results would beobtained for the electric-field to be polarized along the x-z plane).Further introducing normalized notations:

u ₁ =knz ₁ sin² θ₀ , v ₁ =knr ₁ sin θ₀ =kn sin θ₀ {square root}{squareroot over (x₁ ²+y₁ ²)}

u ₂ =knz ₂/sin² θ₀ , v ₂ =knr ₂/sin θ₀ =kn sin θ₀ {square root}{squareroot over (x₂ ²+y₂ ²)}

[0065] where θ₀ is the maximum ray angle relative to the axis of each ofthe objective lenses which serve to focus beam-1 and beam-2, k=2π/λ, andn is the index of refraction of the medium in which beam-1 and beam-2intersect. Note that NA of each objective lens in air is given by sin θ₀(For the sake of simplicity, and without losing any generality, the twoobjective lenses are assumed to have the same NA.)

[0066] It can be shown (e.g., see “Principles of Optics” of Max Born andEmil Wolf, Pergamon Press, 1980, pp.437) that the amplitude at point (x,y, z) associated with beam-1 in the two-beam overlapping region isproportional to U₁ = ∫₀¹J₀(ρ  v₁)^(−j  u₁ρ²/2)ρρ

[0067] Similarly, the amplitude associated with beam-2 at the same point(x, y, z) is proportional toU₂ = ∫₀¹J₀(ρ  v₂)^(−j  u₂ρ²/2)ρρ

[0068] It follows that the “confocal-signal”, resulting from the overlapof beam-1 and beam-2, is proportional to

V=(U ₁ U ₂)²

[0069] FIGS. 8A-8C each display three graphs of normalized“confocal-signal” V/V_(max) plotted as a function of x, y, and zrespectively, where the origin of each graph corresponds with the originO of the x-y-z coordinate system. These graphs are provided by the modelcalculation described above, for several exemplary cases havingdifferent values of the intersecting angle 2α and NA of the objectivelenses. It is assumed that λ=1.3 μm and n=1.35 in all the cases. Adistinct feature prevalent in all the graphs shown in FIGS. 8A-8C isthat V/V_(max) exhibits a Gaussian-like shape in each spatial dimension,diminishing rapidly and monotonically (i.e., there are no additionalside-lobes) with increasing distance from the center of the two-beamoverlapping region. Such characteristics define a sharp resolution inboth the axial as well as transverse directions. In fact, the full widthof each Guassian-like curve at half of its maximum amplitude, D,provides a measure of the spatial extent of the confocal overlappingvolume, hence the spatial resolution, in the given direction for theparticular case in consideration. For comparison purpose, the D valuesobtained for the exemplary cases as well as a few others are listed inTABLE I shown below. TABLE I D (μm) NA α x-direction y-directionz-direction 0.3 30° 1.36 1.18 2.34 0.25 30° 1.63 1.42 2.81 0.2 30° 2.041.77 3.52 0.3 36° 1.46 1.18 1.99 0.2 36° 2.19 1.77 3.00 0.3 45° 1.661.18 1.66 0.2 45° 2.50 1.77 2.50

[0070] Now referring back to FIG. 1A, by rotating about axis 122 at avariable angle φ, scanning mirror 111 is further capable of pivotingillumination beam 117 and observation beam 118 jointly in such a waythat illumination beam 117 and observation beam 118 remain intersectingoptimally at angle θ (=2α) and confocal overlapping volume 121 at theintersection of the two beams moves along an arc-line within object 120,thereby producing an arc-line scan.

[0071] It should be noted that an important characteristic of thearc-line scan described above is that the relative spatial orientationbetween illumination beam 117 and observation beam 118 stays fixed inthe course of the entire scan, once the two beams are arranged tointersect in an optimal manner initially. This is in distinct contrastto the prior art confocal theta scanning systems, where the illuminationand observation beams need to be separately adjusted at each scan point,in order to ensure an optimal intersection. Consequently, the scansperformed by angled-dual-axis confocal scanning head 100 of the presentinvention are inherently of higher precision and faster speed, and arealso less laborious to carry out. Another notable feature ofangled-dual-axis confocal scanning head 100 of the present invention isthat the illumination and observation beam paths can be exchanged,without affecting its performance.

[0072]FIG. 1C shows a second exemplary embodiment of an angled-dual-axisconfocal scanning head of the present invention. In angled-dual-axisconfocal scanning head 150, an illumination reflective focusing element151 is implemented to replace first collimating lens 105, illuminationlens 107, and first mirror 109 in FIG. 1A, providing a dual function offocusing and deflecting illumination beam 117. Likewise, an observationreflective focusing element 152 is used to replace second mirror 110,observation lens 108, and second collimating lens 106 in FIG. 1A,providing a dual function of focusing and deflecting observation beam118. The remainder of angled-dual-axis confocal scanning head 150 sharesthe same components with angled-dual-axis confocal scanning head 100 inFIG. 1A. By way of example, illumination and observation reflectivefocussing elements 151, 152 are in the form of two diffractive lenseswith reflective coatings. The proper design of such reflectivediffraction lenses can be determined by commercially available computermodeling programs and is well known in the art of diffractive lensdesign. Illumination and observation reflective focusing elements 151,152 can alternatively be in the form of curved mirrors. In some cases ofusing curved mirrors such as ellipsoidal mirrors (each having two foci),it is desirable to place fiber ends 101, 102 at the respective firstfocal points of the two ellipsoidal mirrors serving as illumination andobservation focusing elements 151, 152, thereby focusing illuminationbeam 117 and observation beam 118 at the respective second focal pointsof focusing elements 151, 152. All in all, the overall operation ofangled-dual-axis confocal scanning head 150 is similar to the working ofangled-dual-axis confocal scanning head 100, as described above.

[0073]FIG. 1D depicts a simplified schematic illustration of a thirdexemplary embodiment of an angled-dual-axis confocal scanning head ofthe present invention. Angled-dual-axis confocal scanning head 170comprises a first end 171 of a first optical fiber 173 serving as apoint light source; a first end 172 of a second optical fiber 174serving as a point light detector; an angled-dual-axis focusing means inthe form of a lens assembly consisting of first collimating lens 175,second collimating lens 176, and illumination-observation lens 177; anda scanning means in the form of a single scanning mirror 178 having apivoting axis 185.

[0074] In operation, an illumination beam 179 emerges from first end 171of first optical fiber 173 and is directed to first collimating lens175. The collimated beam is then passed onto and focused byillumination-observation lens 177. The focused beam reflects offscanning mirror 178 at first impingement spot 183, and is furtherdirected to a diffraction-limited illumination focal volume (not shownin FIG. 1D) within an object 182. An observation beam 180 emanated froma diffraction-limited, confocal overlapping volume 181 is first receivedby scanning mirror 178 at second impingement spot 184, further passedonto and collimated by illumination-observation lens 177. Observationbeam 180 is then focused by second collimating lens 176 to first end 172of second optical fiber 174. Scanning mirror 178 is positioned such thatillumination beam 179 and observation beam 180 intersect optimally at anangle (θ) within confocal overlapping volume 181, as illustrated in FIG.1B. By rotating about axis 185, scanning mirror 178 is further capableof pivoting illumination beam 179 and observation beam 180 jointly insuch a way that illumination beam 179 and observation beam 180 remainintersecting optimally at angle θ and confocal overlapping volume 181 atthe intersection of the two beams moves along an arc-line within object120, thereby producing an arc-line scan, as in the embodiment of FIG. 1Aor FIG. 1C.

[0075] It should be noted that in an angled-dual-axis arrangement of thepresent invention, as the above exemplary embodiments depict, since theobservation beam is positioned at an angle relative to the illuminationbeam, scattered light along the illumination beam does not easily getpassed into the observation beam, except where the beams overlap. Thissubstantially reduces scattered photon noise in the observation beam,thus enhancing the sensitivity and dynamic range of detection. This isin contrast to the direct coupling of scattered photon noise between theillumination and observation beams in a transmission or reciprocalconfocal microscope, due to the collinear arrangement between the beams.Moreover, by using low NA focusing elements (or lenses) in anangled-dual-axis confocal scanning system of the present invention, theillumination and observation beams do not become overlapping until veryclose to the focus. Such an arrangement prevents scattered light in theillumination beam from directly “jumping” to the observation beam, hencefurther filtering out scattered photon noise in the observation beam.Altogether, the angled-dual-axis confocal system of the presentinvention has much lower noise and is capable of providing much highercontrast when imaging in a scattering medium than the prior art confocalsystems employing high NA lenses, rendering it highly suitable forimaging within biological specimens.

[0076] FIGS. 1A, 1C-1D serve to illustrate only three of manyembodiments of an angled-dual-axis confocal scanning head of the presentinvention. In general, the angled-dual-axis focusing means in anangled-dual-axis confocal scanning head of the present inventioncomprises an assembly of one or more elements for beam focusing,collimating, aligning, and deflecting, as exemplified in FIGS. 1A,1C-1D. Such elements can be in the form of refractive lenses,diffractive lenses, GRIN lenses, focusing gratings, micro-lenses,holographic optical elements, binary lenses, curved mirrors, flatmirrors, prisms and the like. A crucial feature of the angled-dual-axisfocusing means is that it provides an illumination axis and anobservation axis that intersect at an angle, as illustrated in FIG. 1B.The scanning means in an angled-dual-axis confocal scanning head of thepresent invention generally comprises an element selected from the groupconsisting of mirrors, reflectors, acousto-optic deflectors,electro-optic deflectors, mechanical scanning mechanisms, andMicro-Electro-Mechanical-Systems (MEMS) scanning micro-mirrors. A keyfeature is that the scanning means is in the form of a single element,as opposed to two or more separately functioning scanning elements inprior art confocal scanning systems. A preferred choice for the scanningmeans is a flat pivoting mirror, particularly a silicon micro-machinedscanning mirror for its compact and light-weight construction. (Note: toachieve faster scanning, the scanning means can be in the form of twosmaller coplanar pivoting mirrors, such as two silicon micro-machinedscanning mirrors. Owing to their unique fabrication process, thesemirrors can be operated in substantially synchronous motion andconstructed to co-rotate about a common axis so as to scan illuminationand observation beams in a way functionally equivalent to a largersingle scanning mirror.) The fabrication processes of silicon scanningmirrors are described in U.S. Pat. Nos. 6,007,208, 6,057,952, 5,872,880,6,044,705, 5,648,618, 5,969,465 and 5,629,790. The optical fibers in anangled-dual-axis confocal scanning head of the present invention can besingle-mode fibers, multi-mode fibers, birefrigent fibers, polarizationmaintaining fibers and the like. Single-mode fibers are preferable,however, for the ends of single-mode fibers provide a nearly point-likelight source and detector.

[0077] A unique feature of the angled-dual-axis confocal scanning headof the present invention is that the scanning means is placed betweenthe angled-dual-axis focusing means and the object to be examined. Thisenables the best on-axis illumination and observation point-spreadfunctions to be utilized throughout the entire angular range of anarc-line scan, thereby providing greater resolution over a largertransverse field of view, while maintaining diffraction-limitedperformance. Such an arrangement is made possible by taking advantage ofthe longer working distance rendered by using relatively lower NAfocusing elements or lenses in the angled-dual-axis focusing means. Forexample, the present invention uses molded aspherical lenses with NA inthe range of 0.1 to 0.4 that are low cost and readily available in theart. Such lenses have excellent on-axis aberration correction, and aretherefore diffraction-limited for on-axis focusing conditions. Theselenses normally do not have diffraction-limited performance whenfocusing off-axis, and thus cannot be used in confocal scanning systemswhere off-axis performance is important. Such is the case in prior artconfocal scanning systems described in U.S. Pat. Nos. 5,973,828 and6,064,518, where the field of view is limited by the off-axisperformance of objective lenses.

[0078] Moreover, the specific arrangements among various opticalelements and optical fibers in an angled-dual-axis confocal scanninghead can be altered in many ways without deviating from the principleand the scope of the present invention. For instance, the use ofcollimating lenses and beam-aligning mirrors, such as those in FIG. 1Ato help facilitate the shaping and alignment of the illumination andobservation beams, can be optional and vary with the nature of practicalapplications. Other auxiliary optical elements may also be implementedin an angled-dual-axis confocal scanning head of the present invention,to enhance the overall performance. All in all, a skilled artisan willknow how to design an angled-dual-axis confocal scanning head inaccordance with the principle of the present invention, to best suit agiven application.

[0079] By integrating its constituent optical elements on a siliconsubstrate, as exemplified in FIGS. 1A, 1C by way of silicon fabricationtechniques, the angled-dual-axis confocal scanning head of the presentinvention renders a high degree of integrity, maneuverability,scalability, and versatility. Such a design also provides greaterflexibility and higher precision in the alignment of various opticalelements. Although the particular way of making an angled-dual-axisconfocal scanning head of the present invention an integrated device isdictated by the nature of a given application, a silicon substrate isgenerally preferred, for it is well known in the art that variousV-grooves can be etched on silicon in a very precise manner, asdemonstrated in U.S. Pat. No. 6,007,028. The precision of the V-groovesprovides an accurate and secure optical alignment among various opticalelements hosted by these V-grooves, enabling the angled-dual-axisconfocal scanning head thus constructed to be a reliable and modulardevice. Using the embodiment of FIG. 1A as a way of example, mirrors109, 110 can be rotated about their respective axes and translated alongV-grooves 113, 114, respectively, to facilitate the optimal intersectionof illumination and observation beams 117, 118. Illumination andobservation lenses 107, 108 can also be translated along V-grooves 113,114 respectively, to further facilitate the optimal overlapping ofillumination and observation focal volumes 99, 98 as illustrated in FIG.1B. Such alignment procedures can be performed before affixing (e.g., byway of gluing) various optical elements to their respective V-grooves.The scalability and relatively low cost of silicon fabricationtechnology add further advantages to this approach. For example, amicro-optic version of such an integrated angled-dual-axis confocalscanning head can be incorporated in miniature surgical devices,endoscopes, or other in situ devices, suitable for medical applications.

[0080] To provide a two-dimensional scan, an angled-dual-axis confocalscanning head of the present invention can be mechanically coupled to avertical scanning unit, comprising a vertical translation means and acompensation means. The vertical translation means causes theangled-dual-axis confocal scanning head to move toward or away from theobject and hence the illumination and observation beams to probe furtherinto the interior of the object, thereby producing a vertical scan. Atwo-dimensional vertical cross-section scan of the object is thenobtained by assembling a plurality of arc-line scans that progressivelydeepen into the object. The compensation means keeps the optical pathslengths of the illumination and observation beams substantiallyunchanged, thereby ensuring the optimal intersection of the illuminationand observation focal volumes in the course of vertical cross-sectionscans. The combination of the angled-dual-axis confocal scanning headand the corresponding vertical scanning unit constitutes anangled-dual-axis confocal scanning microscope employing verticalcross-section scanning. FIG. 2A depicts an exemplary embodiment of anangled-dual-axis confocal scanning microscope of the present invention.Angled-dual-axis confocal scanning microscope 200, by way of example,comprises an angled-dual-axis confocal scanning head (ADACSH) 201 and amovable carriage 202. For the purpose of illustration, angled-dual-axisconfocal scanning head 201 is in a simplified schematic form of theembodiment shown in FIG. 1A (or FIG. 1C), although any other embodimentsaccording to the present invention can be equivalently utilized. In theembodiment of FIG. 2A, angled-dual-axis confocal scanning head 201 isattached to and further enclosed in movable carriage 202, with opticalfibers 103, 104 extending to the outside of movable carriage 202. Afirst transparent window 203 is mounted on movable carriage 202 forpassage of illumination beam 117 and observation beam 118. Movablecarriage 202 can move up and down in a vertical direction as defined byarrow 204, causing angled-dual-axis confocal scanning head 201 to movetoward or away from object 120 in the process. By doing so, confocaloverlapping volume 121 of illumination beam 117 and observation beam 118further deepen into (or retract from) the interior of object 120,whereby a succession of arc-line scans that progressively deepen intoobject 120 along a vertical cross-section plane 210 is produced, asillustrated by curves 205. The motion of movable carriage 202 can bedriven by a variety of means, for instance, by coupling it to a motor(not shown in FIG. 2A) that is activated by a magnetic, hydraulic,piezoelectric, or other actuator. A skilled artisan can accordinglyimplement a movable stage suitable for a given application.

[0081] Alternatively, to provide a two-dimensional transversecross-sectional scan, a scanning mirror means capable of rotating in twoorthogonal directions can be implemented in an angled-dual-axis confocalscanning microscope of the present invention, such as in one of theexemplary embodiments described above. FIG. 9 shows another exemplaryembodiment of an angled-dual-axis confocal scanning head according tothe present invention. Shown in FIG. 9 is an angled-dual-axis confocalscanning head of the present invention. As a way of example,angled-dual-axis confocal scanning microscope 700 is constructed in away similar to the embodiment of FIG. 1A and hence shares some of theelements in FIG. 1A, as indicated by those identified with the samenumbers.

[0082] Referring to FIG. 9, substituting single scanning mirror 111 inthe embodiment of FIG. 1A is a bi-axial scanning element 320 in the formof a gimbaled assembly of a scanning mirror 304 and a frame 314.Bi-axial scanning element 320 is configured such that scanning mirror304 can rotate about a first pivoting axis 305 (indicated by φ), whereasframe 314 along with scanning mirror 304 can rotate about a secondpivoting axis 315 (indicated by α), thereby providing rotation (φ, α) intwo orthogonal directions. (First and second pivoting axes 305, 315 areconfigured to be substantially orthogonal in this case.) Transversecross-section scans can be accomplished by way of arc-line scanning intwo orthogonal directions using a bi-axial scanning mirror (which pivotsabout two orthogonal axes) as the scanning means of the presentinvention. Such bi-axial scanning mirrors are known in the art, forexample, MEMS type silicon bi-axial scanning mirrors are described inU.S. Pat. Nos. 5,742,419; 6,007,208 and 6,057,952. Also, an aluminumbi-axial scanning mirror using flexure-type hinges is produced byNewport Corporation (Irvine, Calif.) for fast steering of laser beamsabout two orthogonal axes (Newport catalog, page 369).

[0083] Additionally, the vertical scanning unit as described above iscapable of causing the angled-dual-axis confocal scanning head to moverelative to the object being scanned in such a way that a succession oftransverse cross-sectional scans that progressively deepen into theobject can be produced, providing transverse cross-sectional scans atvarying depths within the object. In some cases, where a singletransverse cross-sectional scan is desired at a particular depth, thenthe vertical scanning unit of the present invention can function as ameans of selecting the particular depth of a transverse cross-sectionalscan.

[0084] As illumination beam 117 and observation beam 118 deepen into theinterior of object 120 in the course of vertical scanning, the change intheir respective optical path lengths becomes increasingly large, whichmay cause their respective focal volumes to no longer intersect in anoptimal manner, or even not to intersect at all at the point where thetwo beams physically meet. Furthermore, in interferometry applicationssuch as optical coherence microscopy, the optical path lengths ofillumination beam 117 and observation beam 118 must stay substantiallyfixed in order to generate coherent interference of predominantly singlyscattered light. To maintain the optical path lengths of illuminationbeam 117 and observation beam 118 during vertical scanning, the spacebetween movable carriage 202 and object 120 can be filled with asubstantially transparent fluid 206 having an index of refraction thatis substantially the same as the index of refraction of object 120, suchthat the optical path lengths of illumination beam 117 and observationbeam 118 remain unchanged in the course of vertical scanning. The use ofoptical fibers further provides the necessary flexibility that enablesthe whole assembly of angled-dual-axis confocal scanning head 201 andmovable carriage 202 to move up and down without incurring additionalchange in the optical path lengths of illumination beam 117 andobservation beam 118.

[0085] In the embodiment of FIG. 2A, movable carriage 202, along withangled-dual-axis confocal scanning head 201, is disposed within acontainer 207 filled with fluid 206. An O-ring seal 211 is provided toseal fluid 206 inside container 207, while still permitting movablecarriage 202 to move up and down relative to container 207. Container207 is equipped with a second transparent window 208, in opticalalignment with first transparent window 208 for passage of illuminationand observation beams 117, 118. Container 207 is further connected to afluid injection system 209, serving as a reservoir for replenishingadditional fluid or receiving excess fluid as movable carriage 202 ismoving towards or away from object 120. For imaging of human tissue andother biological samples, fluid 206 can be water, which has an index ofrefraction closely matching that of tissue and biological samples.

[0086] It should be noted that certain aberrations of the illuminationand observation beams may occur as a result of successive passages ofthe beams through first and second transparent windows 203, 208, fluid206, and object 120 in the above embodiment, which may require specificdesigns of the illumination and observation focusing elements that arecorrected for these aberrations. Alternatively, auxiliary opticalelements that are properly designed for correcting such aberrations maybe implemented in the angled-dual-axis focusing means. In most cases ofa converging beam passing through a window or into another object at aninclined angle, the primary aberrations to be corrected for will bespherical aberration, astigmatism, and coma. The magnitude of theseaberrations depend upon many factors, and typically increases with NA ofthe focusing elements, the index of refraction and the thickness of thewindow, and the angle of incidence. The design of suchaberration-corrected focusing elements, or auxiliary optical elementsfor correcting aberrations, can be accomplished by a lens designer ofordinary skill and with the help of an optical design computer programsuch as Zemax™.

[0087] In applications where NA of the focusing elements aresufficiently low and the thicknesses of windows, fluid and objectthrough which the illumination and observation beams successivelytraverse are not large, the aberrations would be small and may not needto be corrected. In such cases, the embodiment shown in FIG. 2A can beutilized, which may incorporate additional remedies for furtherminimizing aberrations. Such remedies include, for example, usingwindows made of Teflon AF or other materials that are transparent andhave an index of refraction closely matching that of water.

[0088] FIB. 2B depicts a second embodiment of an angled-dual-axisconfocal scanning microscope of the present invention, pertaining toapplications where the aforementioned aberrations may not be negligible.In angled-dual-axis confocal scanning microscope 250, a window assemblycomprising two flat transparent windows 254, 255 in an angledarrangement is implemented to replace single flat window 203 in FIG. 2A.The remainder of angled-dual-axis confocal scanning microscope 250shares the same components as angled-dual-axis confocal scanningmicroscope 200 shown in FIG. 2A. The window assembly is designed suchthat illumination axis 97 along with illumination beam 117 andobservation axis 96 along with observation beam 118 (see FIG. 1B) aresubstantially perpendicular to flat windows 255, 254, respectively. Assuch, the window assembly can greatly reduce coma and astigmatism thatwould otherwise be associated with using a single flat window (such aswindow 203 in FIG. 2A). Although spherical aberrations still need to becorrected for in this case, the techniques for making such correctionsare well known in the art of lens design. For instance, a skilledartisan can make use of the design of microscope objectives that arecorrected for glass coverslips of a certain thickness to accomplish thistask.

[0089] For minimizing the aberrations of the illumination andobservation beams, two separate angled windows (254 and 255) and anindex-matching fluid 206 are used as shown in FIG. 2B. Alternatively,these two separate windows may be replaced by a single angle-matchingwindow element, which has first and second window faces in a similarangled arrangement, such that the window faces of the window elementcorrespond to the faces of the two separate windows. As in the case ofusing the two separate windows, the illumination axis should besubstantially perpendicular to the first window face and the observationaxis should be substantially perpendicular to the second window face. Inthis case, this angle-matching window element can be in the form of anoptical prism, which has two of it's faces properly angled anddimensioned to transmit the illumination and observation beamsrespectively with minimum aberrations. For best performance, it is alsopreferable to fabricate the angle-matching prism from a material thathas an index of refraction that most closely matches the fluid. Forexample, the angle-matching prism may be fabricated from Teflon AF™,which has an index of refraction 1.31 that is closely matched to waterhaving an index of refraction 1.33.

[0090] Additional embodiments of the angled-dual-axis confocal scanningmicroscope according to the present invention include replacing theoptical fibers shown in the above embodiments with waveguides (notshown). For example, the first and second optical fibers (illuminationfiber 103 and observation fiber 104, respectively) of the presentinvention can be replaced with first and second optical waveguides(illumination and observation waveguides, respectively).

[0091] In general, the types of first and second optical waveguides usedin an angled-dual-axis confocal scanning head of the present inventionmay be any combination of types depending on the particular applicationthe system is designed for, including, single-mode waveguides,multi-mode waveguides, birefringent waveguides, polarization maintainingwaveguides, buried waveguides, single-mode fibers, multi-mode fibers,birefringent fibers, polarization maintaining fibers, fiber bundles, andthe like. Single-mode waveguides (or fibers) may be preferable for highresolution applications, however, for the ends of single-mode waveguides(or single-mode fibers) provide a nearly point-like light source anddetector. Depending upon the particular application, it may bepreferable to use a mixture of waveguide types and/or fiber types. Forexample, the first fiber (illumination fiber) of the present inventionmay be replaced by any type of single-mode waveguide (or fiber), whichcan provide a point-like light source at the waveguide (or fiber) end,and thus provide a diffraction-limited illumination beam.

[0092] Additionally, the second fiber (observation fiber) of the presentinvention may be replaced by a multi-mode waveguide (or fiber), whichnormally can collect a larger portion of the observation beam with lowerloss than a single-mode waveguide (or fiber). Using this mixture of asingle-mode illumination waveguide (or fiber) and a multimodeobservation waveguide (or fiber) may be a preferred design choice forcases when the observation beam is particularly weak and difficult todetect (such as fluorescence light or light emanating from deep withintissue), or when a more robust design is desired, which has a lowersensitivity to misalignment.

[0093] Other designs are possible where a slit aperture is used at thefirst end (receiving end face) of the multi-mode observation waveguide(or fiber). In this case, a slit aperture on the receiving end face ofthe multi-mode observation waveguide (or fiber) can have the effect ofmaintaining high resolution in one plane (perpendicular to the slitdirection) and allowing less misalignment sensitivity in a secondorthogonal plane (parallel to the slit). It is preferable in most casesto orient the slit in such a way as to maintain the high axialresolution inherent in the design of the dual-angled-axis confocalscanning microscope of the present invention, while allowing moreobservation light to enter the observation fiber and providing a robustdesign.

[0094] For this application, it is possible to fabricate a multimodeobservation waveguide (or fiber) with a slit mask attached to, or formedon the receiving end face. For example, this may be accomplished byattaching a prefabricated slit aperture directly onto the end face withan adhesive or by depositing a layer of metal in the pattern of a slitmask directly onto the end face of the multimode observation waveguide(or fiber).

[0095] All in all, the angled-dual-axis confocal scanning microscope ofthe present invention is designed such that it provides a verticalcross-section scan of an object with enhanced axial resolution, fasterspeed, and larger transverse field of view. Moreover, by moving theangled-dual-axis confocal scanning microscope, or translating theobject, in a transverse direction perpendicular to verticalcross-section plane 210 illustrated in FIG. 2A (or FIG. 2B), a series ofvertical cross-section scans can be taken in a layer-by-layer fashion,which can be assembled to provide a three-dimensional volume image ofthe object.

[0096] For tissue imaging applications, the wavelength of lightgenerally ranges from about 0.8 microns to 1.6 microns, since biologicaltissue samples are particularly transparent in this range. Embodimentsof the angled-dual-axis confocal scanning microscope of the presentinvention are capable of achieving a resolution of about 1-5 microns inthe axial (e.g., the vertical direction shown in FIG. 2A) as well as thetransverse directions, by use of illumination and observation lenseswith NA typically ranging from 0.1 to 0.4, and the intersecting angle θtypically ranging from 45° to 90°. The vertical cross-section scan areais on the order of about 0.5-1 millimeter in both directions. In termsof scanning capabilities, the fast scan rate along an arc-line typicallyranges from 1 to 10 KHz, and the maximum rotation angle (e.g., φ in FIG.1A) from a neutral position of the scanning mirror (e.g., scanningmirror 111 in FIG. 1A) may range from one to several degrees. Generally,the smaller and the lighter the scanning mirror, the faster the scanningrate. For instance, using a silicon micro-machined scanning mirror canprovide scanning rates greater than 10 kHz. The vertical scanning can beperformed at a slower rate of 10-60 Hz, which defines the frame rate ofvertical cross-section scanning and is in the range of video-ratescanning.

[0097] The specific numbers provided above are designed for tissueimaging, to illustrate the utility and the performance of the presentinvention as a way of example. A skilled artisan can utilize the modelcalculation described above to design an angled-dual axis confocalscanning microscope in accordance with the present invention, for agiven application.

[0098]FIG. 3A depicts an exemplary embodiment of a firstangled-dual-axis confocal scanning system of the present invention.Angled-dual-axis confocal scanning system 300 comprises anangled-dual-axis confocal scanning microscope (ADACSM) 301, a lightsource 302, and an optical detector 303. By way of example,angled-dual-axis confocal scanning microscope 301 is in a simplifiedschematic form of one of the embodiments shown in FIGS. 2A-2B. As isapparent to one skilled in the art, other embodiments in accordance withthe present invention can be alternatively implemented. Light source 302is optically coupled to a second end 305 of first optical fiber 103, toprovide an illumination beam to angled-dual-axis confocal scanningmicroscope 301. Optical detector 303 is optically coupled to a secondend 304 of second optical fiber 104, to receive an observation beamcollected by angled-dual-axis confocal scanning microscope 301 fromconfocal overlapping volume 121 within object 120. Light source 302 canbe a fiber laser, a semiconductor optical amplifier, an optical fiberamplifier, a semiconductor laser, a diode-pumped solid state laser, orany other fiber-coupled light source known in the art. The wavelength oflight provided by light source 302 is typically in the range of 0.8-1.6microns, with an average power of about 20 to 200 milliwatts. Lightsource 302 may operate in a continuous wave (CW) or pulsed mode. Opticaldetector 303 can be a PIN diode, or an avalanche photo diode (APD), forinstance. In some cases where the observation beam is weak, aphotomultiplier tube detector may be used. A skilled artisan will knowhow to select a proper optical detector for a given application, asdescribed in “Building Electro-Optical Systems, Making it all work” byPhilip C.D. Hobbs, John Wiley & Sons, 2000. The resulting signals fromoptical detector 303 can subsequently be electronically processed andassembled into corresponding image of vertical cross-section scans ofobject 120 by methods well known in the art. As such, angled-dual-axisconfocal scanning system 300 provides a versatile and high-resolutionimaging device, and can be readily employed in a variety ofapplications. Those skilled in the art can implement thisangled-dual-axis confocal scanning system of the present invention in away suitable for a given application.

[0099] The embodiment described above can also be used to providespecific information pertaining to the polarization state of lightemanated from a polarization-altering, e.g., a birefrigent-scattering,medium. Many biological tissues, such as tendons, muscle, nerve, bone,cartilage and teeth, exhibit birefrigence due to their linear or fibrousstructure. Birefrigence causes the polarization state of light to bealtered (e.g., rotated) in a prescribed manner upon refection. Skin isanother birefrigent medium. Collagen contained in skin is a weaklybirefrigent material. At temperatures between 56-65° C., collagendenatures and loses its birefrigence. Thus, by detecting induced changesin the polarization state of light reflected from a skin sample, animage representing the regions of skin where thermal injury occurs canbe identified. FIG. 3B shows an alternative embodiment of the firstangled-dual-axis confocal scanning system of the present invention,pertaining to applications where polarized light is used to probe abirefrigent-scattering (or other polarization-altering) medium. By wayof example, angled-dual-axis confocal scanning system comprises 350comprises an angled-dual-axis confocal scanning microscope (ADACSM) 351,a polarized light source 352, and a polarizing beamsplitter 353. As inFIG. 3A, angled-dual-axis confocal scanning microscope 351 is in asimplified schematic form of the embodiment shown in FIG. 2, with firstand second optical fibers 103, 104 being polarization maintaining fiberscapable of supporting two orthogonal polarizations. Polarized lightsource 352 is optically coupled to a second end 354 of first opticalfiber 103, to provide an illumination beam 117 with P-polarization toangled-dual-axis confocal scanning microscope 351. Polarizingbeamsplitter 353 is optically coupled to a second end 355 of secondoptical fiber 104, to receive an observation beam 118 with orthogonalP-polarization and S-polarization collected by angled-dual-axis confocalscanning microscope 351 from confocal overlapping volume 121 within abirefrigent-scattering (or other polarization-altering) object 120. Thebirefrigent (or other polarization-altering) “scatterers” in object 120emanate light whose polarization is altered (e.g., rotated) with respectto the polarization of the illumination beam. Such a rotatedpolarization can be represented in two orthogonal components, e.g.,P-polarization and S-polarization. Polarizing beamsplitter 353 separatesP-polarization and S-polarization by routing them to two separateoptical detectors 356, 357 respectively, such that an image representingthese birefrigent (or other polarization-altering) scatterers can beobtained.

[0100] In the embodiment of FIG. 3B, polarized light source 352 can be alaser source, such as a semiconductor optical amplifier, an opticalfiber amplifier, a semiconductor laser, a diode-pumped solid statelaser, or any other fiber-coupled polarized light source known in theart. Polarized light source 352 can also be an assembly consisting of anunpolarized light source optically coupled to a polarizer, such thatpolarized light is produced and transmitted from the assembly toangled-dual-axis confocal scanning microscope 351 as an illuminationbeam. Optical detectors 356, 357 can be PIN diodes, avalanche photodiodes, or photomultiplier tubes. Polarizing beamsplitter 353 can be apolarizing beamsplitter evanescent wave optical fiber coupler, forinstance. As such, angled-dual-axis confocal scanning system 350 issuited for applications in which high resolution and fast imaging ofbirefrigent (or other polarization-altering) media is required. Thoseskilled in the art can implement this angled-dual-axis confocal scanningsystem of the present invention in a way suitable for a givenapplication.

[0101] It should be pointed out that although optical fibers,particularly single-mode fibers, are preferable as optical couplingmeans between various optical elements in this invention, and are usedthroughout this specification wherever optical coupling is called for,other suitable optical coupling means can be alternatively implementedin various angled-dual-axis confocal scanning systems of this invention,without deviating from the principle and the scope of the presentinvention.

[0102]FIG. 4 depicts an exemplary embodiment of an angled-dual-axisconfocal scanning module according to the present invention.Angled-dual-axis confocal scanning module 400 comprises anangled-dual-axis confocal scanning microscope (ADACSM) 401 and athree-port optical circulator 402. For the purpose of illustration,angled-dual-axis confocal scanning microscope 401 is in a simplifiedschematic form of one of the embodiments shown in FIGS. 2A-2B, althoughother embodiments in accordance with the present invention can also beused. Three-port optical circulator 402 is a non-reciprocal device,which couples light incident on port-1 to port-2, and light incident onport-2 to port-3 with high transmission. Whereas light traveling in areversed order encounters high isolation. High isolation also existsbetween port-1 and port-3. For instance, current commercialfiber-coupled circulators known in the art have insertion losses (port-1to 2, and port-2 to 3) less than 0.7 dB, and isolation (port-3 to 2,port-2 to 1) greater than 50 dB. Angled-dual-axis confocal scanningmodule 400 is configured such that port-3-of optical circulator 402 isoptically coupled to a second end 403 of first optical fiber 103; port-1of optical circulator 402 is optically coupled to a second end 404 ofsecond optical fiber 104; and port-2 serves as a bi-directionalinput/output port. In operation, an illumination beam 117 transmitted toport-2 is passed into port-2 of optical circulator 402 and then coupledto fiber end 403 of angled-dual-axis confocal scanning microscope 401 innearly its entirety; and an observation beam 118 collected byangled-dual-axis confocal scanning microscope 401 from confocaloverlapping volume 121 within object 120 is delivered to port-1 viafiber end 404 and then routed to port-2 of optical circulator 402, to befurther utilized or detected in nearly its entirety. As such,angled-dual-axis confocal scanning module 400 of the present inventionprovides a modular, power-efficient, angled-dual-axis confocal scanningdevice with a single input/output port, and can be readily incorporatedin many optical systems, as the following exemplary embodimentsillustrate.

[0103] FIGS. 5A-5B show simplified schematic diagrams of second andthird angled-dual-axis confocal scanning systems incorporating anangled-dual-axis confocal scanning module of the present invention.Depicted In FIG. 5A is the second angled-dual-axis confocal scanningsystem 500, comprising an angled-dual-axis confocal scanning module 501,a self-detecting laser source 502, a third optical fiber 503, and anoptical detector 504. By way of example, angled-dual-axis confocalscanning module 501 is in the form of the embodiment shown in FIG. 4,although any other embodiment in accordance with the present inventioncan be alternatively implemented. Laser source 502 is equipped withfirst output aperture 505 and second output aperture 506. Third opticalfiber 503 is optically coupled to first output aperture 505 of lasersource 502 on one end and to port-2 of angled-dual-axis confocalscanning module 501 on the other, such that an illumination beam 117emitted from first output aperture 505 of laser source 502 istransmitted to angled-dual-axis confocal scanning microscope module 501through port-2, and an observation beam 118 collected byangled-dual-axis confocal scanning module 501 from confocal overlappingvolume 121 within object 120 is in turn back coupled to laser source 502again via first output aperture 505. The feedback into laser source 502of observation beam 118 emanated from object 120 alters the intensity aswell as the modes supported by the laser cavity, and the resultingchanges are detected by optical detector 504 optically coupled to secondoutput aperture 506 of laser source 502. The presence of non-reciprocaloptical circulator 402 in angled-dual-axis confocal scanning module 501enables nearly 100% of illumination beam 117 from laser source 502 to bedelivered to angled-dual-axis confocal scanning microscope 401, as wellas nearly 100% of observation beam 118 collected by angled-dual-axisconfocal scanning microscope 401 to be back coupled to laser source 502,hence maximizing the signal-to-noise ratio in detection. The use ofself-detecting laser source 502, both as a light source and as adetector, further simplifies the structure of this angled-dual-axisconfocal scanning system.

[0104] The self-detecting characteristics of lasers have beenadvantageously exploited in the art to provide an integrated lightsource and detector, as demonstrated in U.S. Pat. Nos. 5,887,009 and5,563,710, by R. Juskaitis et al. in “Semiconductor Laser ConfocalMicroscopy”, Applied optics, 33 (4), pp.578 (1994), and by R. Juskaitiset al. in “Compact confocal interference microscopy”, OpticsCommunications, 109, pp.167 (1994), all incorporated herein byreference. A great deal of effort has also been devoted to eliminatesuch feedback effects; in fact, optical isolators with non-reciprocaloptical elements such as Faraday rotators are designed to eliminate orblock the back-coupling of light. The effects of laser feedback and theresulting perturbations of laser power and frequency depend on manyconditions including laser cavity parameters, and a complete theory isstill lacking at the present time. In the present invention, theself-detecting laser source can be a fiber laser, a semiconductor laser,or a diode pumped solid state laser. A fiber-based laser system, such asthe fiber laser disclosed by the inventors of this application in U.S.Pat. No. 5,887,009, may be used to take advantage of a wide range ofpossible designs and laser cavity parameters to optimize the laserself-detecting properties. A semiconductor laser may also be desirablefor constructing a low cost device.

[0105] Angled-dual-axis confocal scanning system 500 in FIG. 5A furthercomprises a frequency shifting means (or a phase modulator) in the formof frequency shifter 507 optically coupled to third optical fiber 503,such that the frequency of observation beam 118 is shifted before beingback coupled to laser source 502. Frequency shifter 507 can bealternatively coupled to first optical fiber 103, or second opticalfiber 104, for the same purpose of shifting the frequency of observationbeam 118. The feedback of the frequency-shifted (or phase-modulated)observation beam to laser source 502 results in the laser's output beambeing modulated at a beat frequency, thus allowing more sensitiveheterodyne detection.

[0106]FIG. 5B depicts the third angled-dual-axis confocal scanningsystem of the present invention, pertaining to the situation where theself-detecting laser is equipped with only one output aperture.Angled-dual-axis confocal scanning system 550 comprises anangled-dual-axis confocal scanning module 551, a beam-splitting means inthe form of a fiber-optic coupler 552, a self-detecting laser source 553having a single output aperture 554. By way of example, angled-dual-axisconfocal scanning module 551 is in the form of the embodiment shown inFIG. 4, although any other embodiment in accordance with the presentinvention can also be implemented. Angled-dual-axis confocal scanningmodule 551 is optically coupled to fiber-optic coupler 552 by way of athird optical fiber 555, and fiber-optic coupler 552 is in turnoptically coupled to laser source 553. Fiber-optic coupler 552 directs aportion of an output beam emitted from output aperture 554 of lasersource 553 to port-2 of angled-dual-axis confocal scanning module 551,providing an illumination beam 117. An observation beam 118 collected byconfocal scanning module 551 from confocal overlapping volume 121 withinobject 120 is routed back to laser source 553 again via output aperture554. Fiber-optic coupler 552 also routes a remainder of the laser'soutput beam, which carries the perturbations due to the back coupling ofobservation beam 118, to an optical detector 556. To enhance the backcoupling effects, a low-coupling tap coupler, such as a 90/10fiber-optic coupler, is preferred. To make use of heterodyne detection,a frequency shifting means (or a phase modulator) in the form offrequency shifter 557 is optically coupled to third optical fiber 555,such that the frequency of observation beam 118 is shifted before beingback coupled to laser source 553. Frequency shifter 557 can bealternatively coupled to first optical fiber 103, or second opticalfiber 104, for the purpose of shifting the frequency of observation beam118.

[0107] FIGS. 6A-6B depict simplified schematic diagrams of fourth andfifth angled-dual-axis confocal scanning systems of the presentinvention. Shown in FIG. 6A is the fourth angled-dual-axis confocalscanning system 600 of the present invention, comprising anangled-dual-axis confocal scanning module 601 optically coupled to asecond optical circulator 602 by way of a third optical fiber 603, and alight source 604 optically coupled to second optical circulator 602 byway of a forth optical fiber 605. For the purpose of illustration,angled-dual-axis confocal scanning module 601 is in the form of theembodiment shown in FIG. 4, although any other embodiment in accordancewith the present invention can be alternatively implemented. Thirdoptical fiber 603 is optically coupled to port-2 of second opticalcirculator 602 on one end and to port-2 of angled-dual-axis confocalscanning module 601 on the other. Fourth optical fiber 605 in turnoptically couples port-1 of second optical circulator 602 to lightsource 604. In operation, an illumination beam 117 emitted from lightsource 604 is transmitted to port-1 of and in turn passed onto port-2 ofsecond optical circulator 602, and further transmitted to port-2 ofangled-dual-axis confocal scanning module 601. An observation beam 118collected by angled-dual-axis confocal scanning module 601 from confocaloverlapping volume 121 within object 120 is routed back to port-2 of andin turn directed to port-3 of second optical circulator 602, to which anoptical detector 606 is optically coupled.

[0108] In the aforementioned angled-dual-axis confocal scanning system,light source 604 can be a fiber laser, a semiconductor laser, adiode-pumped solid state laser, or a continuous wave or pulsedfiber-coupled light source known in the art. A skilled artisan canimplement an appropriate light source suitable for a given application.Furthermore, the non-reciprocal nature of second optical circulator 602allows nearly 100% of observation beam 118 to be used for detection,thereby maximizing the signal-to-noise ratio.

[0109] The embodiment shown in FIG. 6A can be further modified into aninterferometer configuration, such that the observation beam is combinedwith a portion of the output beam from the light source to createcoherent interference. This can be achieved by inserting abeam-splitting means, such as a fiber-optic coupler or a beamsplitter,between the light source and the second optical circulator in the aboveembodiment. FIG. 6B depicts the fifth angled-dual-axis confocal scanningsystem 650 of the present invention, comprising an angled-dual-axisconfocal scanning module 651, a second optical circulator 652, abeam-splitting means in the form of first fiber-optic coupler 653, and alight source 654. For illustration purposes, angled-dual-axis confocalscanning module 651 is in the form of the embodiment shown in FIG. 4,although any other embodiment in accordance with the present inventioncan also be implemented. A third optical fiber 655 optically couplesport-2 of angled-dual-axis confocal scanning module 651 to port-2 ofsecond optical circulator 652. First fiber-optic coupler 653 is inoptical communication with light source 654 and second opticalcirculator 652, such that it diverts a portion of an output beam fromlight source 654 to port-1 of second optical circulator 652 and aremainder of the output beam to a reference optical fiber 656, therebycreating an illumination beam 117 and reference beam from the sameparent beam. Illumination beam 117 is in turn passed into port-2 ofsecond optical circulator 652, and further transmitted to port-2 ofangled-dual-axis confocal scanning module 651 by way of third opticalfiber 655. An observation beam 118 collected by angled-dual-axisconfocal scanning module 651 from confocal overlapping volume 121 withinobject 120 is routed back to port-2 of and in turn passed onto to port-3of second optical circulator 652, and further directed to a detectionoptical fiber 657. First, second, third, reference, and detection fibers103, 104, 655, 656, 657 have optical path lengths so selected to ensureoptical coherence between the reference and observation beams. Referenceoptical fiber 656 and detection fiber 657 are joined by a secondfiber-optic coupler 658, such that a balanced detection scheme can beemployed for optimizing signal-to-noise ratio in detection.

[0110] To implement balanced detection, a frequency shifting means (or aphase modulator) in the form of frequency shifter 659 is opticallycoupled to detection optical fiber 657 for shifting the frequency of theobservation beam, such that a heterodyne beat frequency is producedbetween the unshifted reference beam and the shifted observation beam atsecond fiber-optic coupler 658 and thus detected by two opticaldetectors 663, 664 at the two outputs of second fiber-optic coupler 658.Frequency shifter 659 can be alternatively coupled to first opticalfiber 103, second optical fiber 104, third optical fiber 655, ordisposed between first fiber-optic coupler 653 and port-1 of secondoptical circulator 652, for the same purpose of shifting the frequencyof the observation beam. Moreover, frequency shifter 659 can beoptically coupled to reference optical fiber 656 for shifting thefrequency of the reference beam, such that a heterodyne beat frequencyis produced between the shifted reference beam and the unshiftedobservation beam at the two outputs of fiber-optic coupler 658. Theunderlying principle of balanced detection and its advantages infiber-optic interferometers are well known in the art, as described byRollins et al. in “Optimal interferometer designs for optical coherencetomography”, Optics Letters, 24(21), pp. 1484 (1999), and by Podoleanuin “Unbalanced versus balanced operation in an optical coherencetomography system”, Applied Optics, 39(1), pp. 173 (2000), incorporatedherein by reference.

[0111] Angled-dual-axis confocal scanning system 650 in FIG. 6B furthercomprises an adjustable optical delay device 660 optically coupled toreference optical fiber 656, serving to maintain coherent interferencebetween the reference and observation beams. Adjustable Optical delaydevice 660 can be alternatively coupled to detection optical fiber 657,or anywhere else along the light path, for achieving the same purpose.In applications where light source 654 has a short coherence length,then delay device 660 can be adjusted such that only single-scatteredlight in observation beam 118 is coherent with the reference beam atsecond fiber-optic coupler 658 and multiple-scattered light, whichtraverses over a larger optical path length in observation beam 118,does not contribute to the coherent interference, therefore providingfurther filtering of multiple-scattered light.

[0112] To further increase the signal-to-noise ratio in detection ofweak optical signals, an optical amplifier 661, such as a two-port fiberamplifier or semiconductor optical amplifier, is optically coupled todetection optical fiber 657 in the embodiment of FIG. 6B, to boost upthe power of the observation beam. Optical amplifier 661 can bealternatively coupled to second optical fiber 104, for the purpose ofamplifying the observation beam. An amplified observation beam allowsfaster scanning rates and consequently higher pixel rates withoutappreciable loss in the signal-to-noise ratio, because a shorterintegration time per pixel of an image is required in data collection.The implementation of balanced detection in this case also allowssubtraction of optical amplifier noise, since most of spontaneousemission of optical amplifier 661 would not occur at the heterodyne beatfrequency described above.

[0113] Light source 654 in FIG. 6B can be an optical fiber amplifier, asemiconductor optical amplifier, a fiber laser, a semiconductor laser, adiode-pumped solid state laser, or a continuous wave or pulsed broadbandOCT light source having a short coherence length, as is well known inthe art. For biological or medical applications, the light source shouldproduce light in the wavelength range of about 0.8 to 1.6 microns, sincebiological tissues are particularly transparent in this range. Thevarious optical fibers are preferably of single-mode type, forsingle-mode fibers offer the advantage of simplicity and automaticassurance of the mutual spatial coherence of the observation andreference beams upon detection. If a polarized light beam is provided bylight source 654, first fiber-optic coupler 653 should be a polarizationmaintaining coupler. Various optical fibers in the system should bepolarization maintaining fibers, capable of supporting two orthogonalpolarizations. First and second optical circulators 402, 652 should alsobe polarization maintaining. In this case, the reference and observationbeams can be brought into the same polarization by rotating eitherreference optical fiber 656, or detection optical fiber 657, beforecoupling it to second fiber-optic coupler 658. Alternatively, apolarization rotation means, such as a Faraday rotator, can be coupledto either reference optical fiber 656, or detection optical fiber 657,such that the reference and observation beams have substantially thesame polarization when combined. Furthermore, second fiber-optic coupler658 should be a polarization maintaining fiber coupler to optimally mixthe polarized observation and reference beams. All in all, by carefullycontrolling the polarizations of the beams in angled-dual-axis confocalscanning system 650, the single-to-noise ratio of detection can beenhanced.

[0114] In the angled-dual-axis confocal scanning systems describedabove, fiber-optic couplers are used to serve as beam-splitting means.Optical fibers, preferably single-mode fibers, are employed for thepurpose of providing optical coupling. Optical circulators arepreferably fiber-coupled circulators. These fiber-optic components,along with the fiber-coupled angled-dual-axis confocal scanning moduleof the present invention, enable the confocal scanning systems thusconstructed to be all fiber-based systems, hence fully exploiting theflexibility, scalability, ruggedness and economical value afforded byoptical fibers.

[0115] Alternatively, those skilled in the art may substitute thefiber-optic couplers and optical circulators by other types ofbeam-splitting and beam-routing means, such as assemblies ofbeamsplitters, prisms, and birefrigent elements, and the optical fibersby other types of free space or bulk optical coupling means well knownin the art, in the angled-dual-axis confocal scanning systems of thepresent invention without deviating from the principle and the scope ofthe present invention. Further, the methods for detection of opticalsignals and for electronic processing of the detected signals intoimages are well known in the art. A skilled artisan can make suitabledesign choices for a given application.

[0116] All in all, the angled-dual-axis confocal scanning microscope ofthe present invention provides many advantages over the prior artsystems, most notably: enhanced axial resolution while maintaining aworkable working distance and a large field of view, fast andhigh-precision scanning, low noise, an integrated and scalablestructure. Moreover, by using low NA focusing elements (or lenses), theangled-dual-axis confocal scanning microscope of the present inventionis capable of minimizing multiple-scattered light, thereby achievinghigher sensitivity and larger dynamic range of detection, a capabilityparticularly desirable for imaging within a scattering medium.Additionally, the integration of the angled-dual-axis confocal scanningmicroscope of the present invention with fiber-optic components and afiber-coupled laser provides an assembly of fiber-based angled-dual-axisconfocal scanning systems that can be particularly powerful tools inbiological and medical imaging applications, such as instruments forperforming optical coherence microscopy and in vivo optical biopsies.

[0117] Although the present invention and its advantages have beendescribed in detail, it should be understood that various changes,substitutions, and alternations can be made herein without departingfrom the principle and the scope of the invention. Accordingly, thescope of the present invention should be determined by the followingclaims and their legal equivalents.

What is claimed is:
 1. An angled-dual-axis confocal scanning systemcomprising an angled-dual-axis confocal scanning head, wherein saidangled-dual-axis confocal scanning head comprises: a) a first opticalwaveguide having first and second ends, for providing an illuminationbeam at said first end; b) a second optical waveguide having first andsecond ends; c) an angled-dual-axis focusing means for focusing saidillumination beam to an illumination focal volume along an illuminationaxis within an object and for receiving an observation beam emanatedfrom an observation focal volume along an observation axis within saidobject such that said observation beam is focused onto said first end ofsaid second optical waveguide; and d ) a bi-axial scanning mirror forproducing a transverse cross-sectional scan, wherein said bi-axialscanning mirror is in optical communication with said focusing means andsaid object, wherein said bi-axial scanning mirror receives saidillumination beam from said angled-dual-axis focusing means and directssaid illumination beam to said illumination focal volume within saidobject, and wherein said bi-axial scanning mirror collects saidobservation beam emanated from said observation focal volume and passessaid observation beam to said angled-dual-axis focusing means; whereinsaid illumination axis and said observation axis intersect at an anglewithin said object, such that said illumination focal volume and saidobservation focal volume intersect at a confocal overlapping volume, andwherein said bi-axial scanning mirror is capable of pivoting saidillumination and observation beams in two orthogonal directions in sucha way that said illumination axis and said observation axis remainintersecting at said angle and that said confocal overlapping volumemoves along a transverse cross-section within said object, therebyproducing said transverse cross-sectional scan.
 2. The angled-dual-axisconfocal scanning system of claim 1 further comprising a verticalscanning unit, wherein said vertical scanning unit comprises: a) atranslation means mechanically coupled to said angled-dual-axis confocalscanning head; and b) a compensation means for ensuring saidintersection of said illumination focal volume and observation focalvolume; wherein said translation means is capable of causing saidangled-dual-axis confocal scanning head to move relative to said object,providing a transverse cross-sectional scan at a selectable depth withinsaid object.
 3. The angled-dual-axis confocal scanning system of claim 2wherein said compensation means comprises a fluid filling a spacebetween said bi-axial scanning mirror and said object, wherein saidfluid is substantially transparent to said illumination beam and saidobservation beam, and wherein said fluid has an index of refraction thatis substantially the same as an index of refraction of said object. 4.The angled-dual-axis confocal scanning system of claim 3 furthercomprising a window means interposed between said bi-axial scanningmirror and said fluid for passage of said illumination and observationbeams.
 5. The angled-dual-axis confocal scanning system of claim 4wherein said window means comprises an index matching transparent flatwindow adjacent to said object, wherein said window has an index ofrefraction that is substantially the same as an index of refraction ofsaid object.
 6. The angled-dual-axis confocal scanning system of claim 4wherein said window means comprises a single angle-matching windowelement having first and second window faces in an angled arrangement,such that said illumination axis is perpendicular to said first windowface and said observation axis is perpendicular to said second windowface, thereby minimizing optical aberrations of said illumination andobservation beams.
 7. The angled-dual-axis confocal scanning system ofclaim 6 wherein said window element is an optical prism comprising saidfirst and second angled window faces, and wherein said prism has anindex of refraction substantially matched to said fluid.
 8. Theangled-dual-axis confocal scanning system of claim 3 wherein said fluidis contained in a sealed hydraulic system, including a reservoir forreplenishing and receiving excess fluid in the course of vertical scans.9. The angled-dual-axis confocal scanning system of claim 1 wherein saidangled-dual-axis focusing means comprises one or more elements selectedfrom the group consisting of refractive lenses, diffractive lenses, GRINlenses, focusing gratings, micro-lenses, holographic optical elements,curved mirrors, and binary lenses.
 10. The angled-dual-axis confocalscanning system of claim 9 wherein said angled-dual-axis focusing meanscomprises a single element, where said element provides saidillumination axis and said observation axis.
 11. The angled-dual-axisconfocal scanning system of claim 9 wherein said angled-dual-axisfocusing means comprises an illumination focusing element and anobservation focusing element, wherein said illumination focusing elementprovides said illumination axis, and wherein said observation focusingelement provides said observation axis.
 12. The angled-dual-axisconfocal scanning system of claim 11 wherein said illumination focusingelement and said observation focusing element are of the same type,comprising a focusing element selected from the group consisting ofrefractive lenses, diffractive lenses, GRIN lenses, micro-lenses, binarylenses, and curved mirrors.
 13. The angled-dual-axis confocal scanningsystem of claim 12 wherein said focusing element has a numericalaperture (NA) in the range of 0.1 and 0.4.
 14. The angled-dual-axisconfocal scanning system of claim 11 further comprising a firstcollimating lens, wherein said first collimating lens receives saidillumination beam from said first end of said first optical waveguideand passes a collimated illumination beam to said illumination focusingelement.
 15. The angled-dual-axis confocal scanning system of claim 14further comprising a second collimating lens, wherein said secondcollimating lens receives said observation beam from said observationfocusing element and focuses said observation beam to said first end ofsaid second optical waveguide.
 16. The angled-dual-axis confocalscanning system of claim 1 wherein said bi-axial scanning mirrorcomprises one or more elements selected from the group consisting ofsilicon scanning mirrors, fast steering mirrors, flexure-type scanningmirrors, and Micro-Electro-Mechanical-Systems (MEMS) scanningmicro-mirrors.
 17. The angled-dual-axis confocal scanning system ofclaim 16 wherein said bi-axial scanning mirror comprises a singlescanning mirror, wherein said scanning mirror is flat and can be pivotedabout two orthogonal axes.
 18. The angled-dual-axis confocal scanningsystem of claim 17 wherein said scanning mirror is a siliconmicro-machined bi-axial scanning mirror.
 19. The angled-dual-axisconfocal scanning system of claim 16 wherein said bi-axial scanningmirror comprises a gimbaled assembly comprising a scanning mirror and aframe, wherein said assembly is configured such that said scanningmirror can rotate relative to said frame about a first pivoting axis andsaid frame along with said scanning mirror can rotate about a secondpivoting axis, thereby providing rotation of said mirror in twoorthogonal directions.
 20. The angled-dual-axis confocal scanning systemof claim 19 wherein said scanning mirror is a silicon micro-machinedscanning mirror.
 21. The angled-dual-axis confocal scanning system ofclaim 1 wherein said angled-dual-axis confocal scanning head ismechanically coupled to a substrate.
 22. The angled-dual-axis confocalscanning system of claim 21 wherein said substrate comprises a siliconsubstrate etched with V-grooves.
 23. The angled-dual-axis confocalscanning system of claim 1 wherein either of said first and secondoptical waveguides comprises an element selected from the groupconsisting of single-mode waveguides, polarization maintainingwaveguides, multi-mode waveguides, birefringent waveguides, single-modefibers, multi-mode fibers, and polarization maintaining fibers.
 24. Theangled-dual-axis confocal scanning system of claim 23 wherein each ofsaid first and second optical waveguides comprises a single-mode opticalfiber.
 25. The angled-dual-axis confocal scanning system of claim 23wherein said second optical waveguide is a multi-mode waveguide, wherebysaid observation beam is focused onto said first end of said multi-modeoptical waveguide, whereby said observation beam is transmitted intosaid multimode waveguide.
 26. The angled-dual-axis confocal scanningsystem of claim 25 wherein said first end of said multimode waveguidecomprises a slit aperture for selecting a portion of said observationbeam such that said portion is first transmitted through said slitaperture before being transmitted into said multi-mode waveguide fordetection at said second end of said multi-mode waveguide.
 27. Theangled-dual-axis confocal scanning system of claim 1 wherein saidobservation beam comprises reflected light emanated from said confocaloverlapping volume within said object.
 28. The angled-dual-axis confocalscanning system of claim 1 wherein said observation beam comprisesfluorescent light emanated from said confocal overlapping volume withinsaid object.
 29. The angled-dual-axis confocal scanning system of claim1 wherein said illumination focal volume and said observation focalvolume are diffraction-limited, determined by main lobes of saidillumination beam's point-spread function and said observation beam'spoint-spread function.
 30. The angled-dual-axis confocal scanning systemof claim 29 wherein said confocal overlapping volume isdiffraction-limited.
 31. The angled-dual-axis confocal scanning systemof claim 26 wherein said multi-mode waveguide is a multi-mode opticalfiber.
 32. The angled-dual-axis confocal scanning system of claim 1further comprising a light source optically coupled to said second endof said first optical waveguide.
 33. The angled-dual-axis confocalscanning system of claim 32 wherein said light source comprises anelement selected from the group consisting of optical fiber amplifiers,fiber lasers, semiconductor optical amplifiers, semiconductor lasers,and diode-pumped solid state lasers.
 34. The angled-dual-axis confocalscanning system of claim 32 wherein said light source comprises apolarized light source, and wherein said second end of said secondoptical waveguide is optically coupled to a polarizing beamsplitter. 35.The angled-dual-axis confocal scanning system of claim 34 furthercomprising two optical detectors, optically coupled to said polarizingbeamsplitter.
 36. The angled-dual-axis confocal scanning system of claim1 further comprising one or more optical detectors optically coupled tosaid second end of said second optical waveguide.
 37. Theangled-dual-axis confocal scanning system of claim 36 wherein said oneor more optical detectors comprise one or more elements selected fromthe group consisting of PIN diodes, avalanche photo diodes, andphotomultiplier tube detectors.
 38. A method of performingangled-dual-axis confocal scanning on a sample, comprising: a)transmitting an illumination beam emerging from a first end of a firstoptical waveguide to an angled-dual-axis focusing means; b) passing saidillumination beam from said angled-dual-axis focusing means to abi-axial scanning mirror; c) using said bi-axial scanning mirror todirect said illumination beam to an illumination focal volume along anillumination axis within said sample; d) using said bi-axial scanningmirror to collect an observation beam emanated from an observation focalvolume along an observation axis within said sample, wherein saidillumination axis and said observation axis intersect at an angle withinsaid sample, such that said illumination focal volume and saidobservation focal volume intersect at a confocal overlapping volume; e)passing said observation beam from said bi-axial scanning mirror to saidangled-dual-axis focusing means; f) focusing said observation beam to afirst end of a second optical waveguide; and g) pivoting said bi-axialscanning mirror in such a way that said illumination axis and saidobservation axis remain intersecting at said angle and that saidconfocal overlapping volume moves along a transverse cross-sectionwithin said sample, thereby producing a transverse cross-sectional scan.39. The method of claim 38 further comprising the step of moving saidfirst ends of said first and second optical waveguides, saidangled-dual-axis focusing means, and said bi-axial scanning mirror inunity relative to said sample and repeating said step of g), whereby asuccession of transverse cross-sectional scans that progressively deepeninto said sample is produced, thereby providing transversecross-sectional scans at varying depths within said sample.
 40. Themethod of claim 39 further comprising the step of filling a spacebetween said bi-axial scanning mirror and said sample with a fluid,wherein said fluid is substantially transparent to said illuminationbeam and said observation beam, and wherein said fluid has an index ofrefraction that is substantially the same as an index of refraction ofsaid sample, for ensuring said intersection of said illumination focalvolume and said observation focal volume in the course of varying thedepth at which transverse cross-sectional scans are produced.
 41. Themethod of claim 40 further comprising the step of disposing a windowmeans between said bi-axial scanning mirror and said fluid for passageof said illumination and observation beams.
 42. The method of claim 40further comprising the step of disposing a transparent window betweensaid fluid and said sample for passage of said illumination andobservation beams.
 43. The method of claim 38 further comprising thestep of mechanically coupling said first ends of said first and secondwaveguides, said angled-dual-axis focusing means, and said bi-axialscanning mirror to a substrate.
 44. The method of claim 38 wherein saidbi-axial scanning mirror comprises a flat scanning mirror that can bepivoted about two orthogonal axes.
 45. The method of claim 44 whereinsaid bi-axial scanning mirror is a silicon micro-machined scanningmirror.
 46. The method of claim 38 wherein said first optical waveguideis a single-mode waveguide, whereby said first end of said single-modewaveguide provides a point source of light, thereby providing saidillumination beam.
 47. The method of claim 38 wherein said secondoptical waveguide is a multi-mode waveguide.
 48. The method of claim 47wherein said first end of said multimode waveguide comprises a slitaperture for selecting a portion of said observation beam such that saidportion is first transmitted through said slit aperture before beingtransmitted into said multi-mode waveguide.
 49. The method of claim 48wherein said slit aperture is a slit mask formed onto said first end ofsaid multimode waveguide.
 50. The method of claim 38 wherein saidangled-dual-axis focusing means comprises a single element, wherein saidelement provides said illumination axis and said observation axis. 51.The method of claim 38 wherein said angled-dual-axis focusing meanscomprises an illumination focusing element and an observation focusingelement, wherein said illumination focusing element provides saidillumination axis, and wherein said observation focusing elementprovides said observation axis.
 52. The method of claim 38 furthercomprising the step of coupling a second end of said first opticalwaveguide to a light source and a second end of said second opticalwaveguide to an optical detector, such that said illumination beam istransmitted from said light source and said observation beam is routedto said optical detector.